Synopsis

Expression of telomerase reverse transcriptase (TERT) is a fundamental hallmark of cancer. Identification of imaging biomarkers of TERT expression will facilitate non-invasive assessment of tumor burden and response to therapy. Our studies in glioma indicate that TERT expression leads to increased redox capacity characterized by elevated ¹H-MRS-detectable glutathione and NADPH. Concomitantly, TERT increases ¹³C-MRS-detectable flux of glucose through the pentose phosphate pathway, which provides NADPH. Importantly, hyperpolarized [U-²H, U-¹³C]-glucose and hyperpolarized [1-¹³C]-dehydroascorbic acid can image these alterations in glucose and redox metabolism. Our study identifies potential non-invasive translational metabolic imaging probes of TERT expression in glioma and possibly other cancers.

Introduction

Methods

Glioma cell models: We examined isogenic immortalized normal human astrocytes without (NHA_pre) and with TERT expression (NHA_post). TERT expression and activity in these models has previously been confirmed².

¹H-MRS: Metabolites were extracted from ~2 x 10⁷ cells by methanol-chloroform extraction and ¹H-MRS spectra acquired on an 11.7T Bruker spectrometer (90°flip angle, 3s relaxation delay, 256 scans) as described³. Principal Component Analysis (PCA) was performed using SIMCA. Discriminating metabolites were identified based on the variable importance in projection (VIP) scores from Partial Least Squares Discriminant Analysis using Metaboanalyst⁴.

Thermally-polarized ¹³C-MRS: Cells were incubated with medium containing 5mM [2-¹³C]-glucose, metabolites extracted and proton-decoupled ¹³C-MRS spectra acquired on an 11.7T Bruker spectrometer (30°flip angle, 3s relaxation delay, 2048 acquisitions) as described⁵.

Hyperpolarized ¹³C-MRS: [U-²H, U-¹³C]-glucose or [1-¹³C]-dehydroascorbic acid were prepared as described^{6, 7}, polarized (HyperSense), dissolved in isotonic buffer and injected into live cells (~10⁸) in a 10mm NMR tube to a final concentration of 10mM. ¹³C-MRS spectra were acquired every 3s for 300s on a Bruker 14.1T spectrometer⁵ and ratios of area under the curve (AUC) for product to substrate quantified.

TERT silencing: Smartpool siRNA against TERT or control siRNA were transfected into NHA_post cells as described⁸.

Statistical analysis: Results are expressed as mean±SD and statistical significance assessed using an unpaired Student’s t-test (* = p<0.05, *** = p<0.005).

Results and Discussion

In order to identify MR-detectable metabolic alterations linked to TERT expression, we investigated glioma models without (NHA_pre) and with TERT expression² (NHA_post, Fig.1A). Unbiased PCA was able to distinguish NHA_pre cells from NHA_post (Fig.1B). Elevated levels of glutathione (GSH, reduced), NAD(P)H, aspartate and AXP (AMP, ADP and ATP) in NHA_post cells relative to NHA_pre were responsible for this discrimination (Fig.1C). These differences were further confirmed by univariate quantification of ¹H-MRS spectra (Fig.1D-1E).

Next, we examined alterations in metabolic fluxes associated with TERT expression. Since glucose flux through the pentose phosphate pathway (PPP) is essential for synthesis of NADPH, which, in turn, maintains GSH in the reduced state, we probed [2-¹³C]-glucose metabolism. PPP fractional flux calculated from the relative proportions of [4-¹³C]-glutamate and [5-¹³C]-glutamate generated from [2-¹³C]-glucose (Fig.2A)⁹ demonstrated that, consistent with elevated NADPH and GSH, PPP fractional flux was higher in NHA_post cells relative to NHA_pre (Fig.2B).

Based on these results, we investigated metabolism of hyperpolarized [U-²H, U-¹³C]-glucose. A representative spectral array showing metabolism of hyperpolarized glucose through the PPP to produce 6-phosphogluconolactone and 6-phosphogluconate (6-PG) in NHA_post cells is shown in Fig.3A. We focused on 6-PG since it has previously been observed in vivo⁷, and found that 6-PG production was higher in NHA_post cells (Fig.3B), suggesting that hyperpolarized [U-²H, U-¹³C]-glucose can non-invasively monitor TERT expression in gliomas.

Since GSH and NADPH levels were higher in NHA_post cells (Fig.1C-1E), we also examined metabolism of hyperpolarized [1-¹³C]-dehydroascorbic acid to ascorbic acid, which was previously shown to provide a readout of GSH and NADPH levels^{6, 10, 11}. Hyperpolarized [1-¹³C]-ascorbic acid production was higher in NHA_post cells relative to NHA_pre (Fig.4A-4B), suggesting that hyperpolarized [1-¹³C]-dehydroascorbic acid can probe TERT expression in gliomas.

In order to link our metabolic imaging biomarkers to TERT expression, we examined NHA_post cells in which TERT expression was silenced by RNA interference (Fig.5A) and compared their metabolism to cells transfected with control siRNA and to NHA_pre cells. As shown in Fig.5B-5C, TERT silencing normalized the ¹H-MRS profile of NHA_post cells resulting in a profile that was indistinguishable from that of NHA_pre cells. Importantly, hyperpolarized [U-²H, U-¹³C]-glucose flux to 6-PG (Fig. 5D) and hyperpolarized [1-¹³C]-dehydroascorbic acid reduction to [1-¹³C]-ascorbic acid (Fig.5E) in NHA_post siTERT cells returned to levels observed in NHA_pre cells, linking our metabolic imaging biomarkers to TERT expression.

Conclusions

Acknowledgements

References

1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell144, 646-674 (2011). doi: 10.1016/j.cell.2011.02.013.

2. Ohba S, et al.Mutant IDH1 Expression Drives TERT Promoter Reactivation as Part of the Cellular Transformation Process. Cancer research76, 6680-6689 (2016). doi: 10.1158/0008-5472.can-16-0696. PMC5290072.

3. Viswanath P, et al.2-hydroxyglutarate-mediated autophagy of the endoplasmic reticulum leads to an unusual downregulation of phospholipid biosynthesis in mutant IDH1 gliomas. Cancer research, (2018). doi: 10.1158/0008-5472.can-17-2926.

4. Xia J, Sinelnikov IV, Han B, Wishart DS. MetaboAnalyst 3.0--making metabolomics more meaningful. Nucleic acids research43, W251-257 (2015). doi: 10.1093/nar/gkv380. PMC4489235.

5. Izquierdo-Garcia JL, et al.IDH1 mutation induces reprogramming of pyruvate metabolism. Cancer research, (2015). doi: 10.1158/0008-5472.can-15-0840.

6. Qin H, et al.Imaging glutathione depletion in the rat brain using ascorbate-derived hyperpolarized MR and PET probes. Sci Rep8, 7928 (2018). doi: 10.1038/s41598-018-26296-6.

7. Rodrigues TB, Serrao EM, Kennedy BW, Hu DE, Kettunen MI, Brindle KM. Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose. Nat Med20, 93-97 (2014). doi.

8. Viswanath P, et al.Mutant IDH1 gliomas downregulate phosphocholine and phosphoethanolamine synthesis in a 2-hydroxyglutarate-dependent manner. Cancer & metabolism6, 3 (2018). doi: 10.1186/s40170-018-0178-3. PMC5881177.

9. Brekke EM, Walls AB, Schousboe A, Waagepetersen HS, Sonnewald U. Quantitative importance of the pentose phosphate pathway determined by incorporation of 13C from [2-13C]- and [3-13C]glucose into TCA cycle intermediates and neurotransmitter amino acids in functionally intact neurons. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism32, 1788-1799 (2012). doi: 10.1038/jcbfm.2012.85. PMC3434630.

10. Bohndiek SE, et al.Hyperpolarized [1-13C]-ascorbic and dehydroascorbic acid: vitamin C as a probe for imaging redox status in vivo. Journal of the American Chemical Society133, 11795-11801 (2011). doi: 10.1021/ja2045925. PMC3144679.

11. Keshari KR, Kurhanewicz J, Bok R, Larson PE, Vigneron DB, Wilson DM. Hyperpolarized 13C dehydroascorbate as an endogenous redox sensor for in vivo metabolic imaging. Proceedings of the National Academy of Sciences of the United States of America108, 18606-18611 (2011). doi: 10.1073/pnas.1106920108. PMC3219134.